1. Field of the Invention
The present invention relates to the separation of enantiomers, i.e., those isomers in which the arrangement of atoms or groups is such that the two molecules are not super-imposable. The invention more particularly relates to a chiral selector useful, for example, as a chiral stationary phase (CSP) in the liquid chromatographic separation (HPLC) of enantiomers of non-steroidal anti-inflammatory agents.
2. Description of the Prior Art
Stereoisomers are those molecules which differ from each other only in the way their atoms are oriented in space. Stereoisomers are generally classified as diastereomers or enantiomers; the latter embracing those which are mirror-images of each other, the former being those which are not. The particular arrangement of atoms that characterize a particular stereoisomer is known as its optical configuration, specified by known sequencing rules as, for example, either + or - (also D or L) and/or R or S.
Though differing only in orientation, the practical effects of stereoisomerism are important. For example, the biological and pharmaceutical activities of many compounds are strongly influenced by the particular configuration involved. Indeed, many compounds are only of widespread utility when employed in a given stereoisomeric form.
Living organisms usually produce only one enantiomer of a pair. Thus only (-)-2-methyl-1-butanol is formed in yeast fermentation of starches; only (+)-lactic acid is formed in the contraction of muscle; fruit juices contain only (-)-malic acid, and only (-)-quinine is obtained from the cinchona tree. In biological systems, stereochemical specificity is the rule rather than the exception, since the catalytic enzymes, which are so important in such systems, are optically active For example, the sugar (+)-glucose plays an important role in animal metabolism and is the basic raw material in the fermentation industry; however, its optical counterpart, or antipode, (-)-glucose, is neither metabolized by animals nor fermented by yeasts. Other examples in this regard include the mold Penicillium glaucum, which will only consume the (+)-enantiomer of an enantiomeric mixture of tartaric acid, leaving the (-)-enantiomer intact. Also, only one stereoisomer of chloromycetin is an antibiotic; and (+)-ephedrine not only does not have any drug activity, but it interferes with the drug activity of its antipode. Finally, in the world of essences, the enantiomer (-)-carvone provides oil of spearmint with its distinctive odor, while its optical counterpart (+)-carvone provides the essence of caraway.
Accordingly, it is desirable and oftentimes essential to separate stereoisomers in order to obtain the useful version of a compound that is optically active.
Separation in this regard is generally not a problem when diastereomers are involved: diastereomers have different physical properties, such as melting points, boiling points, solubilities in a given solvent, densities, refractive indices etc. Hence, diastereomers are normally separated from one another by conventional methods, such as fractional distillation, fractional crystallization, chromatography.
Enantiomers, on the other hand, present a special problem because their physical properties are identical. Thus they cannot as a rule--and especially so when in the form of a racemic mixture--be separated by ordinary methods: not by fractional distillation, because their boiling points are identical; not by conventional crystallization because (unless the solvent is optically active) their solubilities are identical; not by conventional chromatography because (unless the adsorbent is optically active) they are held equally onto the adsorbent. The problem of separating enantiomers is further exacerbated by the fact that conventional synthetic techniques almost always produce a mixture of enantiomers. When a mixture comprises equal amounts of enantiomers having opposite optical configurations, it is called a racemate; separation of a racemate into its respective enantiomers is generally known as a resolution, and is a process of considerable importance.
Various techniques for separating enantiomers are known. Most, however, are directed to small, analytical quantities, meaning that other drawbacks aside, when applied to preparative scale amounts (the milligram to kilogram range) a loss of resolution occurs. Hand separation, the oldest method of resolution, is not only impractical but can almost never be used since racemates seldom form mixtures of crystals recognizable as mirror images.
Another method, known as indirect separation, involves the conversion of a mixture of enantiomers--the racemate--into a mixture of diastereomers. The conversion is accomplished by reacting the enantiomers with an optically pure derivatizing agent. The resultant diastereomers are then separated from one another by taking advantage of their different physical properties. Once separated by, for example, fractional crystallization, or more commonly chromatography, the diastereomers are re-converted back into the corresponding enantiomers, which are now optically pure. Though achieving the requisite separation, the indirect method suffers in that it is time consuming and can require large quantities of optically pure derivatizing agent which can be expensive and is oftentimes not recoverable. Moreover, the de-derivatizing step may itself result in racemization thus defeating the purpose of the separation earlier achieved.
A more current method that avoids some of the drawbacks attendant the indirect method is known as the direct method of separation. The direct method, much like the indirect method, involves the formation of a diastereomeric species. However, unlike the indirect method, this species is transient, with the stability of one species differing from the other.
In one application of the direct method, the mixture of enantiomers is allowed to interact with a chiral stationary phase which, for example, could reside in a chromatographic column. The enantiomer that interacts more strongly with the chiral stationary phase will have a longer residence time, hence a separation of enantiomers will occur. When the mode of interaction with the chiral stationary phase can be characterized, the elution order can be predicted. Examples of chiral stationary phases include those based upon (L)-N-(3,5-dinitrobenzoyl)leucine, which is useful in separating enantiomers of N-aryl derivatized amino acids and esters, and those based upon (L)-N-(1-naphthyl)leucine which has been used to effectively separate N-(3,5-dinitrobenzoyl) derivatized amino compounds. HPLC columns packed with silica-bonded CSP's of a variety of pi-electron acceptors and pi-electron donors--including derivatives of phenylglycine, leucine, naphthylalanine and naphthylleucine are commercially available from Regis Chemical Company, Morton Grove, Ill.
In another application of the direct method, disclosed in copending and commonly assigned patent application Ser. No. 528,007, filed May 23, 1990, now U.S. Pat. No. 5,080,795, enantiomers of such compounds as amino acids, amino esters, alcohols, amines, sulfonic acids or derivatives thereof are separated by means of a liquid membrane containing a chiral carrier, such as the derivatized amino acid (S)-N-(1-naphthyl)leucine octadecyl ester. The chiral carrier is capable of forming a stable complex with one of the enantiomers. The liquid membrane is located on one side of a semi-permeable barrier, and the mixture of enantiomers is located on the other side of the barrier. The liquid membrane containing the chiral carrier impregnates the semi-permeable barrier under conditions effective to permit or cause a stable complex between the chiral carrier and one of the enantiomers to form in the barrier. The liquid membrane containing the stable complex is passed to a second location where the conditions are effective to dissociate the stable complex, allowing the recovery of the complex-forming enantiomer to take place In one embodiment of this application, a hollow fiber membrane is employed as the semi-permeable barrier.
It is widely recognized that stereoisomers of pharmaceutical agents may have drastically different pharmacological potencies or actions. Among those pharmaceutical agents known to elicit differing physiological responses depending on the optical configuration used are the anti-phlogistics, which are those drugs used to counteract inflammation. Antiphlogistics are generally divided into two classes: nonsteroidal anti-inflammatory agents (NSAIAs), which are generally employed in the symptomatic treatment of inflammation, such as occurs with arthritis, and antirheumatics, which act in a more therapeutic fashion.
While NSAIAs can have vastly different chemical structures, most are aryl acidic molecules (though the acidic function is not essential for anti-inflammatory activity) or metabolic precursors thereof, often possessing two to three aromatic or heteroaromatic rings, either fused or linear and which are often nonplanar; the presence of a halogen or isostere atom or group usually enhances activity. Nonsteroidal anti-inflammatory agents are generally categorized into the following groups: 1) salicylates, which are derivatives of salicylic acid and include agents such as aspirin; 2) 5-pyrazolone derivatives, most of which are 3,5-pyrazolidinedione derivatives and include agents such as phenylbutazone; 3) fenamates and isosteres, which are either n-arylanthranilic or 2-aminonicotinic acid derivatives, which include such agents as meclofenmate sodium; 4) oxicams, which are mostly N-heterocyclic carboxamides of 4-hydroxy-2H-1,2-benzothiazine 1,1-dioxide, and include such agents as piroxicam; 5) other acidic compounds, such as aminobenzoic acid salts (e.g. aminobenzoate potassium), pyrocathecol derivatives (e.g. nepitrin) and the sulfonanilide derivative, nimesulide; and 6) nonacidic heterocyclic compounds, such as indazole derivatives.
The more commonly employed NSAIAs, however, are those in the category known as arylacetic acid compounds. Most arylacetic acid compounds share certain structural features: a carboxyl group or its equivalent, such as enolic acid, hydroxamic acid, sulfonamide, or a tetrazole moiety, separated by one carbon atom from a planar aromatic nucleus (hence making them acidic molecules). To the flat aromatic nucleus, one or more large lipophilic groups may be attached. The presence of an .alpha.-methyl substituent normally enhances potency, while an increase in size of this .alpha.-substituent usually diminishes activity.
Generally, the category known as arylacetic acid compounds is subdivided into the following subgroups: phenylacetic acid compounds, such as diclofenac sodium; phenylpropionic acid compounds, such as ibuprofen and naproxen; phenylbutyric acid compounds, such as indobufen; aryloxyalkanoic acid compounds, such as furobufen; and heteroarylacetic acid compounds, such as etodolac.
Given the importance of nonsteroidal anti-inflammatory agents, arylacetic acid compounds in particular, and of the criticality of employing the proper enantiomeric form, much effort has been made investigating methods for obtaining the desired optical configuration of these compounds. These techniques have ranged from attempts at synthesizing one of the two optical isomers in pure form in the first instance, to tailoring the more traditional methods of enantiomer separation to meet this particular need.
While asymmetric synthesis would theoretically reduce or eliminate the need for complex stereospecific separations, these synthetic techniques have met with limited commercial success in general applications, and have had even less success with respect to nonsteroidal anti-inflammatory agents. Stereochemical isolation and purification of these compounds have thus far relied upon chromatographic separation techniques.
Of the separation techniques in this regard, most have required derivatization of the particular NSAIA involved with some techniques even employing a chiral derivatizing agent so as to form diastereomers. However, these derivatization-dependent methods introduce increased time and cost factors into the separation and, even more importantly, introduce the possibility of error into the separation process, which now requires further steps and reactions to achieve resolution, which itself may prove to be ultimately less effective.
Procedures which require derivatization of NSAIAs with chiral reagents in order to obtain diastereomers are particularly problem prone, given that the rate of reaction of the chiral reagent with individual enantiomers may be different thus leading to a ratio of diastereomers which does not reflect the initial ratio of analyte enantiomers. Further, these procedures require scrupulous maintenance of the enantiomeric purity of the chiral reagent and avoidance of partial racemization of the analyte and chiral reagent during the course of the derivatization procedure. Lastly, the diastereomeric product ultimately obtained may give non-identical detection results, thus requiring additional validation steps.
Accordingly, to avoid these drawbacks, efforts have been made to obtain direct chromatographic separation of underivatized NSAIA enantiomers. However, these efforts have failed to be generally applicable to the entire class of NSAIAs, and have failed to be even generally applicable to any category of NSAIAs, such as the arylacetic acid category, which includes naproxen and other profen-type agents. For example, Hermansson and Eriksson in "Direct Liquid Chromatographic Resolution of Acidic Drugs Using Chiral .alpha.-1-Acid Glycoprotein Column (Enantiopac.RTM.), J. Liq. Chromatogr., 9, 621 (1986) report a chromatographic separation factor (.alpha.) of greater than 4 for underivatized naproxen using an .alpha.-1-acid glycoprotein chiral stationary phase and an achiral ion pairing reagent. However, the separation of enantiomers using a protein-derived stationary phase suffers from several important drawbacks which make these methods less than desirable for practical applications: First, since proteins are only available in one enantiomeric form, or antipode, elution orders cannot be reversed, which is often desirable in the analytical determination of enantiomeric purity. And, in any event, if a chiral selector is to find practical application as an enantioselective membrane transport agent, it is desirable that it be available in both enantiomeric forms. Secondly, proteins and protein-derived chiral stationary phases typically have rather low stability compared to synthetic chiral selectors, thus the lifetime of a protein selector or a chiral stationary phase derived therefrom, will not be as great as that for a synthetic selector; this is especially true where elevated temperatures, extremes of pH or organic solvents are involved. Finally, owing to the extremely low concentration of binding sites on the protein, preparative scale resolutions are not feasible.
Other efforts in this regard include that reported by Petterson and Gioeli in "Improved Resolution of Enantiomers of Naproxen by the Simultaneous Use of a Chiral Stationary Phase and a Chiral Additive in the Mobile Phase", J. Chromatogr., 435, 225 (1988) in which underivatized enantiomers of naproxen were separated on a quinidine-based chiral stationary phase using quinine as a mobile phase additive. Separation, however, was marginal, with .alpha.=1.18, and poor band shape was exhibited, hence making this technique impractical for preparative purposes.
Thus there continues to be a pressing need for a process of separating enantiomers of NSAIAs, especially those categorized as arylacetic acid compounds, which does not require derivatization and which is not protein-based and which can provide a high degree of resolution and is generally applicable at least across an entire category of NSAIAs.